*Target (protein/gene name): NAD-dependent glyceraldehyde-3-phosphate dehydrogenase *NCBI Gene # or RefSeq#:RefSeq: hp2017_0904 *Protein ID (NP or XP #) or Wolbachia#:RefSeq: ADZ50007.1 *Organism (including strain):Helicobacter pylori Etiologic Risk Group (see link below):Group I—Pathogens Newly Recognized in the Past Two Decades *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Helicobacter pylori is a gram-negative bacterium responsible for about 80% of stomach ulcers and 90% of ulcers in the duodenum. Part of its name is derived from its spiral shape which has been determined to help the bacteria penetrate the stomach's protective mucous lining. The bacteria produce substances that weaken the mucous lining. This causes the stomach to be more susceptible to gastric acids. Another way the bacteria damages the stomach is attaching to the cells of the stomach and stimulating the production of excess stomach acid. Recently, this bacteria has been a significant problem in the US. About 20% of people under 40 and about 50% of the adults over 60 years old are infected. These rates are higher in developing countries. However, being infected by the bacteria does not necessarily mean that one will develop ulcers or stomach cancer. It is still unclear why this happens.
Essentiality of this protein: Glyceraldehyde-3-phosphate dehydrogenase is essential for evasion from neutrophils. http://www.ncbi.nlm.nih.gov/pubmed/16565520 Complex of proteins?: Yes Druggable Target: No
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
<span style="background-color: #ffffff; color: #222222; font-size: 12px;">Query 1 MKIFINGFGRIGRCVLRAILERNDTNPQLEVIGINDPANWEILAYLLEHDSVHGLLNKEA 60
+K+ INGFGRIGR V RA L+ NP +EV+ +ND LA+LL++DSVHG L+ E
Sbjct 2 VKVGINGFGRIGRNVFRAALK----NPDIEVVAVNDTGGANTLAHLLKYDSVHGRLDAEV 57
Query 61 RYSNGKLIIGSLEIPVFNSIKDLK-------GVGVIIECSGKFLEPKTLENYLLLGAKKV 113
+ L++ EI + + +D + GV +++E +G+F + + +L GAKKV
Sbjct 58 SVNGNNLVVNGKEI-IVKAERDPENLAWGEIGVDIVVESTGRFTKREDAAKHLEAGAKKV 116
Query 114 LLSAPFMGEYDEKQYPTLVYGVNHFLYQNQA--IVSNASCTTNAIAPICAILDKAFSIKE 171
++SAP E T+V GVN Y +A ++SNASCTTN +AP +L + F I
Sbjct 117 IISAPAKNED-----ITIVMGVNQDKYDPKAHHVISNASCTTNCLAPFAKVLHEQFGIVR 171
Query 172 GMLTTIHSYTSDQKLIDLAHPLDKRRSRAAASNIIPTTTKAALALHKVLPNLKNKMHGHS 231
GM+TT+HSYT+DQ+++D +H D RR+RAAA +IIPTTT AA A+ VLP LK K++G +
Sbjct 172 GMMTTVHSYTNDQRILDASHK-DLRRARAAAESIIPTTTGAAKAVALVLPELKGKLNGMA 230
Query 232 VRVPSLDVSMIDLSLFLEKKALKESINDLLITASKGTLKGVLEIDLKERVSSDFISNPNS 291
+RVP+ +VS++DL LEK+ E +N L A++G LKG+L + VS D+ + S
Sbjct 231 MRVPTPNVSVVDLVAELEKEVTVEEVNAALKAAAEGELKGILAYSEEPLVSRDYNGSTVS 290
Query 292 VIIASDLTFTLEN-MVKIMGWYDNEWGYSNRLVDMAQFM 329
I + T ++ MVK++ WYDNE GYS+R+VD+A ++
Sbjct 291 STIDALSTMVIDGKMVKVVSWYDNETGYSHRVVDLAAYI 329</span>
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 99%
---- Max % Identities:43%
---- % Positives 63%
---- Chain used for homology: O
Purification Method: The protein was purified from E.coli by conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. http://www.ncbi.nlm.nih.gov/pubmed/12841648 Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids 332 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 37.072 Molar Extinction coefficient of your protein at 280 nm wavelength: 34630 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*GC% Content for gene: 51% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: RefSeq: hp2017_0904
*Protein ID (NP or XP #) or Wolbachia#: RefSeq: ADZ50007.1
*Organism (including strain): Helicobacter pylori
Etiologic Risk Group (see link below): Group I—Pathogens Newly Recognized in the Past Two Decades
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Helicobacter pylori is a gram-negative bacterium responsible for about 80% of stomach ulcers and 90% of ulcers in the duodenum. Part of its name is derived from its spiral shape which has been determined to help the bacteria penetrate the stomach's protective mucous lining. The bacteria produce substances that weaken the mucous lining. This causes the stomach to be more susceptible to gastric acids. Another way the bacteria damages the stomach is attaching to the cells of the stomach and stimulating the production of excess stomach acid. Recently, this bacteria has been a significant problem in the US. About 20% of people under 40 and about 50% of the adults over 60 years old are infected. These rates are higher in developing countries. However, being infected by the bacteria does not necessarily mean that one will develop ulcers or stomach cancer. It is still unclear why this happens.
Essentiality of this protein: Glyceraldehyde-3-phosphate dehydrogenase is essential for evasion from neutrophils.
http://www.ncbi.nlm.nih.gov/pubmed/16565520
Complex of proteins?: Yes
Druggable Target: No
*EC#: 1.2.1.12
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/Mol/reaction-popup.php4?id=29386&type=I&displayType=marvin
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glyceraldehyde3phosdehydr1.pdf
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glyceraldehyde3phosdehydr1.pdf
-- link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glyceraldehyde3phosdehydr1.pdf
-- List cost and quantity of substrate reagents and supplier
Triethanolamine Buffer-$31, 3-phosphoglyceric acid disodium salt - $13.80, L-cysteine hydrochloride - $22.40
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
<span style="background-color: #ffffff; color: #222222; font-size: 12px;">Query 1 MKIFINGFGRIGRCVLRAILERNDTNPQLEVIGINDPANWEILAYLLEHDSVHGLLNKEA 60 +K+ INGFGRIGR V RA L+ NP +EV+ +ND LA+LL++DSVHG L+ E Sbjct 2 VKVGINGFGRIGRNVFRAALK----NPDIEVVAVNDTGGANTLAHLLKYDSVHGRLDAEV 57 Query 61 RYSNGKLIIGSLEIPVFNSIKDLK-------GVGVIIECSGKFLEPKTLENYLLLGAKKV 113 + L++ EI + + +D + GV +++E +G+F + + +L GAKKV Sbjct 58 SVNGNNLVVNGKEI-IVKAERDPENLAWGEIGVDIVVESTGRFTKREDAAKHLEAGAKKV 116 Query 114 LLSAPFMGEYDEKQYPTLVYGVNHFLYQNQA--IVSNASCTTNAIAPICAILDKAFSIKE 171 ++SAP E T+V GVN Y +A ++SNASCTTN +AP +L + F I Sbjct 117 IISAPAKNED-----ITIVMGVNQDKYDPKAHHVISNASCTTNCLAPFAKVLHEQFGIVR 171 Query 172 GMLTTIHSYTSDQKLIDLAHPLDKRRSRAAASNIIPTTTKAALALHKVLPNLKNKMHGHS 231 GM+TT+HSYT+DQ+++D +H D RR+RAAA +IIPTTT AA A+ VLP LK K++G + Sbjct 172 GMMTTVHSYTNDQRILDASHK-DLRRARAAAESIIPTTTGAAKAVALVLPELKGKLNGMA 230 Query 232 VRVPSLDVSMIDLSLFLEKKALKESINDLLITASKGTLKGVLEIDLKERVSSDFISNPNS 291 +RVP+ +VS++DL LEK+ E +N L A++G LKG+L + VS D+ + S Sbjct 231 MRVPTPNVSVVDLVAELEKEVTVEEVNAALKAAAEGELKGILAYSEEPLVSRDYNGSTVS 290 Query 292 VIIASDLTFTLEN-MVKIMGWYDNEWGYSNRLVDMAQFM 329 I + T ++ MVK++ WYDNE GYS+R+VD+A ++ Sbjct 291 STIDALSTMVIDGKMVKVVSWYDNETGYSHRVVDLAAYI 329</span>---- Show pairwise alignment of your BLASTP search in NCBI against the PDB---- Query Coverage: 99%
---- Max % Identities:43%
---- % Positives 63%
---- Chain used for homology: O
Current Inhibitors: Tetrose diphosphate http://www.jbc.org/content/234/10/2510.full.pdf
Expression Information (has it been expressed in bacterial cells): The protein was expressed in E. Coli cells
http://www.ncbi.nlm.nih.gov/pubmed/3049151
Purification Method:
The protein was purified from E.coli by conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography.
http://www.ncbi.nlm.nih.gov/pubmed/12841648
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids
332
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
37.072
Molar Extinction coefficient of your protein at 280 nm wavelength: 34630
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
CDS Gene Sequence (paste as text only):
*GC% Content for gene: 51%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References:
http://www.webmd.com/digestive-disorders/h-pylori-helicobacter-pylori